Glycated (glycosylated) hemoglobins have gained acceptance as a relevant index of long-term blood glucose control in patients with diabetes mellitus. As used subsequently in this specification the term glycated hemoglobin refers to relatively stable condensation products of hemoglobin with glucose (and possibly glucose phosphates), as compared with more labile hemoglobin-glucose adducts, supposedly of the aldimine (Schiff base) type and generated by a non-enzymatic reaction between glucose and amino groups of hemoglobin. The latter are believed to be converted into the stable (formerly termed "glycosylated") type an Amadori rearrangement (cf. M. Roth: Clin.Chem. 29 (1983) 1991).
Glycated hemoglobin A components were first recognized when hemoglobin A was subjected to electrophoresis and cation exchange chromatography. Owing to their more negative charge and consequently higher electrophoretic migration rates towards the anode than that of the major component hemoglobin A (HbA.sub.o) they were named the "fast" hemoglobins (HbA.sub.1). The fast hemoglobins constitute a series of minor hemoglobins among which inter alia HbA.sub.1a, HbA.sub.1b and HbA.sub.1c have been identified according to their differential migration rates. Of these HbA.sub.1c is present in greatest quantity in erythrocytes both from normal subjects and from diabetic patients. HbA.sub.1c is known to be glycated at the N-terminal. valine of the .beta.-chains of hemoglobin A. However, recent studies have indicated that glycation may also occur at the amino group of lysine side chains and that all hemoglobins, including HbA.sub.o and HbA.sub.1c, may comprise such glycated sites. The labile (aldimine) precursor of HbA.sub.1c (usually referred to as "pre-HbA.sub.1c ") is not encompassed by the above definition of HbA.sub.1c.
It is now generally accepted that the level of HbA.sub.1c in a blood sample is a good index for the individual's glycemic control. Normal adults have about 90% of their total hemoglobin A as HbA.sub.o and 3-6% as HbA.sub.1c, the balance consisting of other minor hemoglobins including HbA.sub.1a and HbA.sub.1b. However, the level of HbA.sub.1c in patients with type 1 (juvenile) and type 2 (maturity-onset) diabetes ranges from about 6% to about 15%. The quantification of the HbA.sub.1c level in diabetic patients is regarded as a useful means of assessing the adequacy of diabetes control, in that such measurements represent time-averaged values for blood glucose over the preceding 2-4 months (cf. J. S. Schwartz et al.: Annals of Intern. Med. 101 (1984) 710-713).
The ideal laboratory method for measuring HbA.sub.1c should be specific (e.g. not influenced by the presence of pre-HbA.sub.1c) accurate, precise, easily standardized, inexpensive and facile. Unfortunately, the methods currently available such as cation exchange chromatography, high performance liquid chromatography (HPLC), affinity chromatography or electrophoresis (isoelectric focusing) do not meet all of these criteria simultaneously. For a general review of these methods and their attempted implementation for diabetes control, reference is made to J. S. Schwartz et al., supra.
The direct measurement of glycated hemoglobin based on immunological methods has been suggested in the prior art. In this respect reference is made inter alia to British Patent No. 1.580.318 and to U.S. Pat. No. 4.478.744. The method devised in the former requires comparatively large amounts of human HbA.sub.1c for immunization and the latter makes use of a laboriously synthesized peptide for preparation of the antigen. In both instances the immunoassay is conducted with animal antiserum or fractions thereof containing so-called polyclonal antibodies. Such polyclonal antibodies are difficult to prepare in a reproducible manner and are generally not regarded as sufficiently specific for the assay of HbA.sub.1c in a highly complex mixture of closely related hemoglobin molecules.
It is the object of the present invention to overcome the drawbacks of the methods known heretofore for laboratory measurements of the extent of glycation, of hemoglobin A. The invention is based on the surprising observation that it is possible by using appropriate immunization, screening and selection procedures to isolate cell lines (hybridomas) which produce monoclonal antibodies that will bind specifically to epitopes containing glycated amino groups of hemoglobin, such as that of the N-terminal valine of the .beta.-chains of hemoglobin A, whereas substantially less or essentially no binding occurs to the corresponding non-glycated site.
In particular, the generation of antibodies recognizing the glycated N-terminal valine residues of the HbA.sub.1c .beta.-chains is a surprising achievement in view of the fact that models of the hemoglobin molecule based on X-ray crystallographic studies indicate that these residues, at least in their non-glycated state, are buried in a central cavity of the molecule and hence would be expected to be difficultly accessible to such large molecules as immunoglobulin antibodies. This cavity encompasses the so-called 2,3-diphosphoglycerate (DPG)-pocket wherein the much smaller DPG molecule is trapped between the N-terminal ends of the two, .beta.-chains of deoxyhemoglobin. When the .beta.-chains come closer together in oxyhemoglobin, the DPG molecule is pushed out of its binding pocket. For illustration, see R. E. Dickerson and I. Geis in "Hemoglobin: Structure, Function, Evolution, and Pathology" p. 40 (The Benjamin/Cummings Publ. Co., 1983).